Previous publications have suggested that commonly prescribed antidepressants have the potential to stimulate the proliferation of extant tumors in human and rodent in vivo and in vitro models. The direct effects of amitriptyline and fluoxetine were evaluated in assays that detect different aspects of proliferative responses at pharmacologically relevant drug concentrations. Three in vitro assays of cellular proliferation and clonal growth were used with human (MCF7, PA-1 and LS174T) and murine (B16.f10, C-3 and B16.f1) tumor cell lines. The cells were exposed to amitriptyline or fluoxetine (0.001-100 microM) for different time periods (1-7 days) and at varying serum concentrations (0.1-15%). Amitriptyline and fluoxetine failed to significantly stimulate tumor cell proliferation, DNA synthesis, or colony formation. Both drugs inhibited B16.f10 colony growth at concentrations above 5 microM along with significant suppression of DNA synthesis in B16.f10 and C-3 cells at 30 microM. Although there were generally no effects on cell proliferation by the drugs in the microtiter tetrazolium assay, several rare instances of stimulation were noted. Amitriptyline and fluoxetine were consistent in their lack of effect or inhibition with the human or murine tumor cell lines in conventional in vitro assays of cell proliferation and clonogenicity in optimal or suboptimal culture conditions.